Cholera continues to be a major problem in developing countries worldwide. While the current licensed vaccine may be promising for some populations, it is not effective for all populations. Reactogenicity has been an obstacle in the development of a live, attenuated cholera vaccine. Several reports indicate that an inflammatory component may cause reactogenicity, prompting our lab to explore the ability of vaccine strains to induce IL-8. Initial findings indicate a trypsin- and proteinase K-sensitive, but heat-insensitive IL-8- inducing factor in culture supernatants. In this proposal, we intend to identify and characterize the supernatant component(s) responsible for IL-8 induction, which we hypothesize may contribute to the reactogenicity of cholera vaccine strains. Using flagellin mutants and possibly chromatography, we propose to identify the factor(s) that causes IL-8 secretion in human intestinal epithelial cells. Through the use of Western blots, chemical inhibitors, electrophoretic mobility shift assays, and reporter constructs, we propose to identify the signaling pathways involved in IL-8 expression. Lastly, we propose to construct a cholera vaccine strain that lacks the IL-8 stimulatory component(s), and may therefore have reduced reactogenicity. [unreadable] [unreadable] [unreadable]